Herpesviruses are important human pathogens that result in life-long persistent infections with numerous clinical manifestations. Herpes simplex virus (HSV) is among the most frequently encountered pathogen by the general population. Thus understanding the biology of this virus is important in the development of efficacious treatments of these infections. Our goal is to understand the mechanism by which the virus acquires its infectious coat and the role of the virus encoded functions in this pathway, primarily by analyzing the functions of two tegument proteins, the UL36 and UL37 gene products. Our hypothesis is that the UL36 and UL37 gene products specify essential and unique functions for the maturation of the assembled particle into an infectious virion. The goals of this proposal are to understand how these two proteins function in the morphogenesis of the infectious particle, identification of the functional domains required for these activities and the interactions that occur during this process. This could potentially lead to the discovery of novel pathways that can be targeted by antiviral intervention. The specific aims to achieve these goals are: Specific Aim 1: Investigating the role of UL36 and UL37 in the maturation of the virus particle using mutant viruses. UL36 may act to translocate capsids to the cytoplasmic site for final envelopment, whereas, UL37 is required for Golgi stabilization prior to the arrival of capsids at this site. Light microscopy, electron microscopy and immuno-EM assays will be used to determine the association of UL36 mutant virus particles with the cellular cytoskeleton and analyze axonal transport in primary neurons. The Golgi structure will be analyzed in the presence and absence of UL37 and the contribution of UL36, capsid assembly and cis-acting signals for facilitating Golgi stabilization will be determined. Specific Aim 2: Protein-protein interactions of UL36 and UL37 with virus and cellular proteins. Protein-protein interactions are critical for a variety of viral and cellular processes. The UL36 and UL37 encoded polypeptides will be used as probes to identify protein-protein interactions using a combination of cell biology (cellular localization), genetic (yeast two-hybrid), biochemical (protein pull-down) assays. Specific Aim 3: Structural studies of UL36 and UL37 in capsids and the mature virion. A capsid binding assay and cryo-electron tomography imaging of virions will illuminate the association of these proteins with a multi-protein assembly (capsid/virion). Structural data is important for understanding how these proteins function in virus egress. Specific Aim 4: Identification of functional domains of UL36 and UL37. Transposon and site-directed mutagenesis will be used to identify functional domains of the UL36 and UL37 genes. Genetic complementation assays will be used to screen the mutants for functional activity. Mutations that specify lethal defects in function will be introduced into the virus using a marker-rescue/marker-transfer method. The outcome will be a functional map of UL36 and UL37 that identifies domains important for capsid translocation, Golgi stabilization, Golgi trafficking, virion incorporation and protein-protein interactions This analysis will identify domains of the proteins that have the potential to be used as targets for antiviral intervention